Beta-lactamases hydrolyze the amide bonds of the beta-lactam ring of sensitive penicillins and cephalosporins, are widely distributed among microorganisms, and play an important role in microbial resistance to beta-lactam antibiotics. Several methods for detecting the presence of microbial beta-lactamase have been developed. For example, chemical methods for the detection of the enzymatic hydrolysis of the beta-lactam ring include: (a) the acidimetric method, which employs a pH color indicator to detect the decrease in pH resulting from the formation of a new carboxyl group; (b) the iodometric method, which is based on the decolorization of a starch-iodine complex by the end products of beta-lactamase hydrolysis, which act as reducing agents to reduce iodine in the complex; and (c) the chromogenic cephalosporin method, which is based on a color change following the hydrolysis of a chromogenic cephalosporin substrate (R. B. Sykes and K. Bush, "Physiology, Biochemistry and Inactivation of Beta-lactamases," Chemistry and Biology of Beta-lactam Antibiotics 3: 155-207 (1982) R. B. Morin and M. Gorman (eds.), Academic Press, New York. An alternative to the chemical methods is a microbiological assay method which is based on the loss of antibacterial activity following the hydrolysis of the beta-lactam ring.
Microbial acylases which remove the acyl side chains of susceptible penicillins or cephalosporins are also produced by many microorganisms. The cleavage of acyl side chains from beta-lactam antibiotics often results in a decrease in pH and reduction of antibiotic activity. Acidimetric and microbiological methods may not differentiate .beta.-lactamase activity from acylase activity.
Although microbial beta-lactamases do not act exclusively on penicillins or on cephalosporins, many show a predominance of penicillinase or cephalosporinase activity. Thus, chemical or microbiological methods which utilize a single beta-lactam substrate cannot differentiate penicillinase activity from cephalosporinase activity and often give a false, negative result for beta-lactamase activity.
There exists a need for a method which differentiates penicillinase activity from cephalosporinase activity and distinguishes between beta-lactamase and acylase activity.
The aforementioned co-pending U.S. patent application discloses and claims a method for detecting the presence of beta-lactamase activity from microbial sources using a fluorescence assay. It is desirable to provide means to enhance the fluorescent signal generated in said assay in order to increase the sensitivity of the assay. Additionally, it is desirable to provide improved methodologies for decreasing the time of the assay. The prior art teaches the use of a buffered solution containing formaldehyde in order to enhance fluorescent development of said assay (Taylor, D. N., K. C. S. Chen, K. Panikabutra, C. Wongba, A. Chitwarkern, P. Echeverria, and K. K. Holmes, Lancet ii: 625-626 (1985)). This use of a formaldehyde solution as a fluorescence developer was described in a subsequent article by K. C. S. Chen and K. K. Holmes (J. Clin. Microb. 23: 539-544 (1986)).
Despite the increase in sensitivity achieved by the formaldehyde developer solution, there exists a need for greater sensitivity in order to expand the applicability of the assay. The present invention fulfills this need.